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The Journal of Immunology, 2004, 172: 4133-4141.
Copyright © 2004 by The American Association of Immunologists

Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center1

Zeljka Korade Mirnics2,*, Eva Caudell{dagger}, YanHua Gao*, Kazuhiko Kuwahara{ddagger},§, Nobuo Sakaguchi{ddagger}, Tomohiro Kurosaki, Joan Burnside||, Károly Mirnics2,# and Seth J. Corey2,*,{dagger}

* Department of Pediatrics, University of Pittsburgh, School of Medicine, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213; {dagger} Division of Pediatrics, University of Texas-M. D. Anderson Cancer Center, Houston, TX 77030; {ddagger} Department of Immunology, Graduate School of Medical Sciences, Kumamoto University School of Medicine, Honjo, Kumamoto, Japan; § PRESTO, Japan Science and Technology Corporation, Kawaguchi, Japan; Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan; || Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711; and # Departments of Psychiatry and Neurobiology, PittArray Microarray Core, University of Pittsburgh, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-{kappa}B, elongation factor 1{alpha}, CD79b, octamer binding factor 1, Ig H chain, stathmin, and {gamma}-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn->PU.1->GANP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stimulation of the B cell receptor (BCR)3 activates signaling cascades that lead to proliferation, differentiation, and effector functions (1, 2, 3). BCR activation recruits members of nonreceptor protein tyrosine kinases (PTKs) of the Src, Syk, Tec, and Csk families (3, 4, 5, 6). BCR evoked tyrosine phosphorylation of Lyn, and Syk triggers a biochemical cascade in which a variety of cellular proteins become tyrosine phosphorylated (3, 7).

Although native B cell express several Src family PTKs (Lyn and Blk), the chicken B cell DT40 expresses only Lyn (8). Src PTKs are believed to have overlapping expression and redundant function (9). However, because Lyn is the only Src PTK family member expressed in DT40 cells, this cell line provides a unique experimental system to study Lyn’s role in B cell signaling. A unique feature of DT40 cells is their ability to undergo efficient homologous recombination, so that more than several dozen genes have been inactivated and resulting lines intensively studied (10). Lyn-deficient DT40 cells show a defective BCR cross-linking response, manifested by slow calcium mobilization and profoundly reduced phosphorylation/activation of Syk (11). Lyn-deficient cells also show increased sensitivity to apoptosis; however, precise molecular deficiencies have not yet been associated with the Lyn-/- phenotype.

Lyn-deficient mice have a dramatic phenotype that resembles systemic lupus erythematosus in humans (12, 13). The B cells of these mice show reduced response to BCR cross-linking or LPS activation (14). With age, these mice develop high serum IgM levels, lymphadenopathy, and splenomegaly (15). In contrast, deficiency of other Src PTK family members expressed predominantly within B cells (Blk, Hck, Fgr, and Lck) (16, 17, 18) does not produce a prominent phenotype (reviewed in Ref. 19). Although Lyn-deficient mice show normal B cell development within the bone marrow, the number of peripheral B cells in spleen, mesenteric lymph nodes, and Peyer’s patches is reduced (15). Lyn deficiency is also associated with an increased number of immature B cells in the periphery (12, 15).

Recent advances in functional genomics highlighted the advantages of investigating complex gene expression patterns for the discovery of novel signaling and disease-related pathways (20, 21, 22, 23). To determine putative downstream effectors associated with Lyn signaling in B cells, we compared Lyn-/- and wild-type avian DT40 cells using custom-made chicken cDNA membrane arrays (23). The analysis revealed a set of genes involved in B cell development and signaling. One of these is germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), a B cell differentiation Ag. Expression of GANP, a 210-kDa phosphoprotein, is up-regulated in germinal centers of Ag-immunized murine spleens (24). Through its C terminus, GANP binds to MCM3, a component of the DNA replication licensing system that regulates initiation of DNA replication (25). CD40 stimulation induces a phosphorylation (Ser502)-dependent primase activity in the central region of GANP (26). Thus, GANP likely regulates DNA replication in activated centrocytes. A yeast two-hybrid screen identified G5PR as a binding partner for GANP, and because G5PR can recruit phosphatases PP5 and PP2A, the GANP/MCM3 complex may be regulated by phosphorylation during cell cycle progression (27). GANP expression may be partly regulated by the transcription factor PU.1, because the GANP promoter has a PU.1 consensus sequence (28). Interestingly, Lyn and PU.1 are found to be critical for both B cell and myeloid development and signaling. In this study, we show that GANP and PU.1 expression are deficient in Lyn-deficient avian DT40 cells and Lyn-deficient murine splenocytes. GANP promoter activity was dependent on both Lyn and the PU.1-sensitive site. Together, these data suggest the presence of a Lyn->PU.1->GANP pathway in B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and Lyn-deficient mice

DT40 cell lines were generated by Dr. T. Kurosaki’s laboratory as previously described (11). Lyn-/- mice were obtained from Drs. C. Lowell (University of California, San Francisco, CA) (15) and D. Linnekin (National Cancer Institute, Frederick, MD) (29).

Cell cultures

Wild-type and Lyn-deficient DT40 cells were grown in RPMI 1640 medium with 10% FBS, 1% chicken serum, penicillin/streptomycin, and 50 µM 2-ME as described previously (11). Cells were harvested 24 h after plating at a density of 2 x 105/ml. Approximately 108 cells were used for isolation of total RNA and mRNA.

cDNA array experiments

Nitrocellulose cDNA arrays. The cDNAs on the arrays were selected from a chicken activated T cell library (30). Sequence data and information regarding the identity of the clones can be found at the University of Delaware Chick EST Project website (http://chickest.udel.edu). Inserts were amplified by PCR using vector primers, and the quality and quantity of PCR products were evaluated by agarose gel electrophoresis (30).

Isolation of RNA, labeling, and hybridization procedures. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, and mRNA extraction was performed using the Poly(A) Track mRNA isolation kit (Promega, Madison, WI). A total of 1 µg of mRNA was used to perform first-strand cDNA synthesis using [{alpha}-32P]dCTP in an oligo(dT)-primed reverse transcription reaction (Life Technologies, Rockville, MD). A total of 20,000,000 cpm of radioactively labeled first-strand cDNA in 5-ml volume was hybridized overnight to each of the membranes. The membranes were washed, exposed to phosphor screens, and scanned (Molecular Dynamics, Sunnyvale, CA). Hybridizations were repeated three times, using wild-type or Lyn-deficient DT40 cultures, respectively, as starting material. After each of the hybridizations, the membrane arrays were stripped, sealed, and exposed to x-ray film for 48 h. None of the stripped membrane arrays demonstrated residual radioactivity. Reused membranes did not show sensitivity differences between subsequent hybridizations (data not shown).

Image acquiring and analysis. TIF files were analyzed using GeneSight 4.0 software (BioDiscovery, Marina Del Rey, CA). After defining the metagrid and subgrids, the spot pattern was inspected and manually adjusted for pin performance and membrane stretch. For each probe spot, mean/median signal intensity and mean/median local background measurements were performed. Spots were considered for further analysis if they produced a signal from >40% of the spot area. Array measurements were imported into Excel 2000 (Microsoft, Redmond, WA) and GeneSpring 4.1 (Silicon Genetics, Redwood City, CA) for further analysis.

Data analysis. Data were normalized across the cDNA arrays. The 50th percentile of all measurements was used as a positive control for each sample; each measurement for each gene was divided by this synthetic positive control, assuming that this ratio was at least 10. This synthetic control was the median of the gene’s expression values over all of the samples. The bottom 10th percentile was used as a test for correct background subtraction. The duplicate spots had to be within 40% of each other in intensity to be considered for further analysis. First, for each gene, the intensities of duplicate spots were averaged. Then, the averages were cross-compared in a 3 x 3 matrix, resulting in nine comparative values of balanced differential expression (BDE) for each of the 1440 genes. Based on the nine BDEs, we generated a p value and performed a standard Z-score transformation (BDE gene - average BDE/SD). Finally, the nine BDE values were averaged, determining the combined differential expression value for each gene. Genes were considered to show differential expression between conditions if they 1) showed an increased or decreased BDE value across seven of nine comparisons, 2) fell outside of 2 SDs from the mean combined differential expression value, and 3) reported a Z-score that exceeded 2 SDs from the mean in either direction. Hierarchical clustering was performed using Cluster/Treeview software available from Stanford University (Palo Alto, CA). BDE values were log transformed, and the genes (but not the experiments) were clustered as described by Eisen et al. (31) using the complete clustering method.

Northern hybridization

Total RNA was isolated from the cell lines and mouse spleen using TRIzol (Life Technologies). The hybridization was performed using the NorthernMax system (Ambion, Austin, TX). Briefly, 20 µg of total RNA was loaded on a formamide gel, electrophoresed at 5 V/cm, transferred to a Bright Star nylon membrane (Ambion), and cross-linked by UV light exposure. Chicken probes were generated by enzymatic digestion of the original clones made in Dr. J. Burnside’s laboratory. Mouse probes were generated using gene-specific primers in a standard PCR using normal spleen cDNA as a template. The product was labeled using a Random Primed DNA labeling kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. To demonstrate comparable RNA loading, a 404-bp murine GAPDH probe was used. The 620-bp probe for chicken PU.1 was obtained by PCR of chicken cDNA from DT40 cells, with the forward primer, 5'-GAAGGATTTCCCCTCATTCC-3', and the reverse primer, 5'-TGCGATTGCCTTTCTGTATG-3'. Northern blot was performed on total RNA.

Isolation of mouse spleen B cells, immunization of mice, and immunohistochemistry

Mouse spleen B cells were enriched and cultured in RPMI 1640 medium as described previously (24, 32). Mice were immunized by multiple i.p. injections with SRBC (Colorado Serum, Denver, CO) as described previously (24). Immunohistochemistry was done using Abs directed against GANP or phospho-Ser502-GANP as described previously (33).

Western blot

Wild-type and lyn-/- mice were immunized with SRBC (Cappel, West Chester, PA) by i.p. injections. Mice were sacrificed 7 days postinjection, and spleens were harvested and snap frozen in methyl butane. Spleens were homogenized in 500 µl of lysis buffer (1% Nonidet P-40, 25 mM Tris (pH 7.5), 140 mM NaCl, 10 mM NaF, 5 mM EDTA, protease inhibitor mixture, and 1 mM Na vanadate) with a PowerGen 125 homogenizer to prepare whole-cell lysates. After centrifugation for 30 min at 13,000 rpm at 4°C, supernatants were obtained and a modified Bradford protein assay (Bio-Rad, Hercules, CA) was performed. A total of 150 µg of protein lysate per sample was loaded and separated by SDS-PAGE on a 12.5% gel, transferred onto polyvinylidene difluoride membrane, and probed for PU.1 (1:250), Lyn (1:1000), or Actin (1:1000) (all Abs from Santa Cruz Biotechnology, Santa Cruz, CA).

GANP promoter reporter assay

The pGV-P constructs containing wild-type and PU.1/-134 mutated GANP promoter sites have been described elsewhere (28). DT40 and Lyn-deficient DT40 cells were cotransfected in triplicate with 10 µg of GANP promoter constructs and 500 ng of pRLTK (Promega) containing the Renilla luciferase gene as an internal control. The Dual-Luciferase Reporter Assay system (Promega) was used following the manufacturer’s protocol. Cells were harvested ~42 h posttransfection, pelleted, and washed once with PBS, before being lysed with passive lysis buffer. The supernatant was used to measure the luciferase activity using Analytical Luminescence Laboratory Monolight 2010 Luminometer. A total of 20 µl of cell lysate was added to 100 µl of reagent LARII, and luciferase activity was measured. To measure the Renilla luciferase activity, 100 µl of stop and glow buffer (Promega) was added to same tube. As a positive control, cells were transfected with vector pGL2 control (Promega) containing the luciferase gene under SV40 promoter element. To compare the activity of the same promoters in wild-type and Lyn-deficient cell lines, the Renilla luciferase activity (internal control) was used to normalize the luciferase activity values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lyn-/- DT40 cells demonstrate consistent gene expression differences

To determine the signaling pathways in which Lyn is involved, we have studied a chicken DT40 B cell line with a deletion of the Lyn gene (11). In contrast to the Src kinases expressed in normal B cells, wild-type DT40 (DT40WT) cells express only Lyn PTK. As a result, in the DT40 system, other Src PTKs family members could not compensate for the absence of Lyn.

Comparisons between the DT40WT and DT40Lyn-/- cells revealed gene expression differences over the full range of signal intensity (data not shown); many of the transcript changes were identifiable even by visual inspection. The duplicate probe spots of the cDNA arrays showed consistent performance within all experiments for >99% of the genes investigated. The two cultured cell lines did not show apparent growth rate or morphological differences, and, as expected, the majority of genes reported comparable expression between the two cell lines.

We identified 45 genes, the expression of which was decreased in Lyn-/- DT40 cells relative to DT40WT cells, whereas only 2 genes with increased expression fulfilled the same criteria (Tables I and II). Interestingly, many of the 45 genes with decreased expression were related to B cell response, regulation of transcription, proliferation, or cytoskeletal organization.


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  		Table I. Significantly changed genes between the Lyn+/+ and Lyn-/- DT40 cell lines: those decreased in the Lyn-deficient DT40 cell line

 

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  		Table II. Significantly changed genes between the Lyn+/+ and Lyn-/- DT40 cell lines: those increased in the Lyn-deficient DT40 line

 
To uncover gene expression patterns across the comparisons, we used hierarchical clustering. Clustering the log values of BDEs across the nine comparisons revealed that most of our consistently changed genes showed up in a confined region of a cluster map (Fig. 1). Importantly, several different probes associated with the genes elongation factor 1{alpha} (EF1{alpha}) and CD74 were clustered as nearest neighbors, providing us with a further validation of our experimental strategy. In addition to genes from Tables I and II, many of the genes found in the two clusters did not fulfill the criteria we set forth for determining the most changed genes. These genes belonged to the classes of B cell response (MHC class I, {alpha}2-HS glycoprotein) and proliferation (TGF, crn, 3-hydroxyacyl-CoA dehydrogenase, zinc finger protein). Some of these expression differences likely represent biologically significant changes that are difficult to distinguish from experimental noise. Fig. 2 depicts expression microarray demonstrated differences for 10 selected genes across the nine DT40 cell comparisons. These genes were selected to represent the whole range of changed genes shown in Table I (CD74, CD22, GANP, NF-{kappa}B, EF1{alpha}, transglutaminase, OBF-1, Ig HC, stathmin, prothymosin, and {gamma}-actin). In addition, we included two genes (MRJ of DNAJ, L5 ribosomal subunit) that showed consistently decreased expression that did not meet our statistical criteria for inclusion in Tables I and II. Using a new isolate of wild-type and Lyn-/- DT40 cells, we verified the changes in gene expression for all of these genes by Northern hybridization (data not shown).



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  		FIGURE 1. Hierarchical clustering of Lyn-/- and Lyn+/+ gene expression differences. The right panel dendrogram is generated using the cDNA array comparison data in Cluster/Treeview by the method of Eisen et al. (31 ). Note that several regions show clustering of consistent increases and decreases between the two compared conditions (Lyn+/+ vs Lyn-/-). Two such regions, depicted by color bars, are enlarged: the left panel represents clusters of genes that reported consistently decreased expression in the Lyn-deficient cell lines, whereas the middle panel corresponds to clusters encompassing genes that reported the most consistent increases. Note that, within the left panel, the software clustered three different probes of EF1{alpha} and three MHC class II genes as nearest neighbors, further demonstrating the power of this analytical approach.

 


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  		FIGURE 2. Differential expression of 13 selected genes. Logged BDE values are plotted on the y-axis across the nine comparisons (C1–C9) on x-axis. Note that most of the comparisons report consistent decrease for these 13 genes.

 
Lyn-/- mouse spleen and Lyn-/- DT40 cells exhibit reduced GANP expression

We hypothesized that some Lyn functions are conserved across species and experimental systems, and that a portion of the observed gene expression changes in the Lyn-/- DT40 cells will be observed in Lyn-/- mice. To test this hypothesis, we have chosen to examine the expression of five genes (GANP, PHB2, transglutaminase, pyruvate kinase, and EF1{alpha}) in the spleen total RNA of the Lyn-/- mice. Besides their differential expression in Lyn-/- DT40 and DT40WT cells, these genes were chosen based upon their importance for B cell signaling or for their unknown role in the Lyn signaling cascade. Using total RNA isolated from Lyn-deficient mouse spleen and Northern hybridization, we were able to verify expression changes for GANP, but not for EF1{alpha}, pyruvate kinase, and transglutaminase (data not shown). A Northern blot performed showed that GANP expression was markedly reduced in murine splenocytes from Lyn-deficient mice (Fig. 3).



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  		FIGURE 3. Northern blot of murine splenocytes for GANP expression. Splenocytes were purified from lyn-/- mice or their homozygous wild-type littermates. Total RNA was harvested, and Northern blotting was performed for expression of murine GANP, with GAPDH cDNA used as a control for RNA loaded.

 
GANP-dependent proliferation is absent in the Lyn-/- mice

Although GANP has not been previously implicated in Lyn signaling, it is involved in proliferation of B cells and formation of germinal centers (24). Interestingly, Lyn-deficient mice show decreased proliferation of peripheral B cells and poorly formed germinal centers within mesenteric lymph nodes and spleen (12, 15). These findings, combined with our data, suggested that GANP expression within splenic B cells might be at least partially responsible for the phenotype of the Lyn-deficient mice. Because the expression level of GANP is low in normal B cells, we performed SRBC stimulation of control and Lyn-deficient mice to induce expression of GANP (24). As expected, staining of spleen sections with biotinylated reagent of peanut agglutinin (24) in wild-type animals showed formation of giant germinal centers with strongly labeled GANP+ cells (Fig. 4). However, spleen sections obtained from Lyn-/- mice showed absence of germinal centers and GANP staining (Fig. 4A). An Ab raised against the serine phosphorylated form of GANP showed the same staining pattern (Fig. 4B). In wild-type mice, CD40 stimulation is known to result in GANP up-regulation in germinal centers (24). To test whether this mechanism is preserved in Lyn-deficient splenic B cells, we stimulated B cells with anti-CD40 Ab. CD40 activation leads to rapid activation of Lyn (24, 34, 35). This stimulation led to a characteristic proliferative response in wild-type cells, whereas proliferation was almost completely absent in Lyn-/- mouse B cells (Fig. 5). The inability to up-regulate GANP and produce a proliferative B cell response in Lyn-deficient B cells suggests the existence of a Lyn PTK-dependent, GANP+ cell population, which may be essential for germinal center formation and/or proliferation.



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  		FIGURE 4. Lyn-/- mouse germinal centers stimulated by SRBC fail to induce GANP expression. A, GANP staining. Sections of Lyn-/- and Lyn+/- mouse spleen germinal centers stained with peanut agglutinin (PNA) (top) and anti-GANP Ab (bottom) after SRBC stimulation. Note that heterozygous mice (left panels) proliferated and up-regulated GANP, whereas in Lyn-deficient mice (right panels), this response was absent (RP, red pulp; WP, white pulp; GC, germinal center). B, Phospho-Ser502-GANP staining. Sections were stained for Ab against the serine phosphorylated form of GANP, as described elsewhere (24 ).

 


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  		FIGURE 5. Lyn-/- and Lyn+/- B cells respond differently to CD40 stimulation. Anti-GANP staining of unstimulated and CD40-stimulated B cells from heterozygous and Lyn-/- mice. Note that Lyn-deficient B cells do not up-regulate GANP in response to CD40 stimulation.

 
PU.1 expression and activity is Lyn PTK dependent

Because GANP expression is dependent on PU.1 activity, the effect of Lyn on PU.1 was studied. Northern blot analysis shows that PU.1 expression was greater in wild-type than Lyn-deficient DT40 cells or Lyn-deficient murine splenocytes (Fig. 6, A and B). Differences in PU.1 transcription factor activity between wild-type and Lyn-deficient cells were also seen in luciferase promoter assay. The promoter for GANP (-134) containing the luciferase reporter construct into wild-type and Lyn-deficient DT40 cells. A 2-fold greater activity in wild-type cells was observed. Confirming that GANP promoter activity was at least partly due to its Lyn-sensitive PU.1 binding site, we observed that wild-type enhanced activity was reduced by 67% when the PU.1 binding site was mutated (Fig. 6C). Interestingly, PU.1-sensitive luciferase activity was diminished by the same degree in Lyn-deficient DT40 cells. Together, these studies suggest that Lyn affects GANP expression by enhancing both expression and activity of PU.1.



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  		FIGURE 6. Lyn regulates level and activity of the transcription factor PU.1. A, Northern blot analysis of PU.1 in DT40 cells. Comparison of wild-type and Lyn-deficient DT40 cells show that there are decreased levels of PU.1 in Lyn-deficient cells compared with wild-type cells. B, Western blot analysis of PU.1 in murine splenocytes. Comparison of wild-type and Lyn-deficient murine splenocytes show that there are decreased protein levels of PU.1 (indicated by arrow) in Lyn-deficient cells. In lower panel, absence of Lyn is demonstrated in Lyn-deficient murine splenocytes. Comparable levels of protein loading are shown by actin blotting in the middle panel. Protein lysates were prepared from splenocytes from individual mice (indicated by number). As controls, protein lysates were prepared from the murine cell lines Ba/F3 and 32D. C, GANP promoter activity in wild-type and Lyn-deficient DT40 cells. Wild-type and Lyn-deficient DT40 cells were transfected with constructs containing the GANP promoter and the luciferase reporter gene. Wild-type and Lyn-deficient cells were also transfected with the construct containing a mutated PU.1 binding site (*PU.1), as described elsewhere. Values are expressed as relative luciferase units (mean ± SD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lyn is involved in multiple signaling processes. We used cDNA array analysis to identify previously unknown Lyn-dependent functional signaling cascades that are conserved across species and experimental systems. This experimental design helped us to assess the overall involvement of Lyn in B cell signaling, as well as to identify a subset of signaling events that uniquely depend on preserved Lyn function.

The changes identified in the Lyn-/- DT40 cells argue that Lyn PTK is involved in a multitude of cellular processes including BCR-signaling (CD74, CD22, Ig {alpha} H chain, Ig-associated {beta}, and CD79b), proliferation (GANP, tumor-associated MAGE like, nucleoside diphosphate kinase, stathmin, prothymosin, and transglutaminase), control of transcription (transcription factors, EF1{alpha}, cleavage and polyadenylation specificity factor, CEBP, TCF1, OBF-1, ICS BP), immunity and inflammation response (NF-{kappa}B), and cytoskeletal organization ({gamma}-actin, transglutaminase, chaperonin CCT {delta}, stathmin).

Interestingly, compared with the number of genes that are down-regulated in the absence of Lyn, there are only a few genes that showed consistently increased expression as a result of Lyn deficiency. This is somewhat surprising because Src PTKs can activate or inhibit immunoreceptor-mediated signal transduction depending on the substrates they phosphorylate (36, 37, 38). However, we measured steady-state changes in unstimulated DT40 cells, and gene expression increases as a result of Lyn deficiency may become obvious only when these cells are activated. However, despite the enormous discovery potential of high-throughput transcriptome profiling, general limitations of the microarray datasets include both type I and type II errors. In our experimental series, for the most robustly changed genes, type I error was limited, and the critical data were verified in follow-up experiments. However, we expect that there are further (and potentially significant) expression changes that were not uncovered by our analysis (type II error). Furthermore, many important biological processes occur at the protein level, and these events were not the focus of our investigation.

Lyn deficiency leads to altered expression of genes forming the BCR complex

We found significantly altered expression of at least five genes that are involved in the formation of BCR complex. Ig H chain is the major subunit of Ag-binding component, CD79b is the intracellular signaling component of the BCR, CD22 is a BCR coreceptor, whereas OBF-1 is a transcription factor (39). Although it is well established that Lyn phosphorylates CD79b and CD22, it has not been reported that Lyn affects their expression (40). Furthermore, CD74 functions as a chaperone, transporting subunits of the BCR to the plasma membrane. These decrements in the expression of multiple related genes are highly suggestive of an impaired regulation/assembly of the BCR and may result in an altered BCR signaling. OBF-1 is a transcription factor, most abundantly expressed in B cells (41). Mice deficient in OBF-1 have reduced numbers of mature B cells, a severe reduction in the number of recirculating B cells, and poor germinal center formation (42). Altogether, these data suggest that Lyn controls BCR expression, assembly, and signaling in part through OBF-1 (4, 43). Lyn has been described as a double-edged kinase that can promote or inhibit growth (14). B cell apoptosis can be induced through CD40/CD40 ligand or through BCR cross-linking (44, 45). Lyn-mediated phosphorylation of CD22 also serves to control growth. These processes may also occur simultaneously or sequentially, reducing B cell viability in a complex process. Thus, this microarray analysis suggests that a PTK can affect a molecule through both posttranslational and transcriptional means.

Some Lyn PTK functions may not be unique

Not all of the gene expression changes described above need be uniquely dependent on preserved Lyn function. Although DT40 cells do not express Src kinases other then Lyn (11), both DT40 cell line and mammalian B cells contain BTK and Syk (46). We hypothesize that gene expression differences found in Lyn-/- DT40 cells but not in the mouse Lyn-deficient spleen isolate (e.g., EF1{alpha}, transglutaminase) are due to the redundant effects of other Src kinase members. This redundancy is clearly present in B cells, although it may not be characteristic of other cell populations and tissues in the Lyn-deficient mice. The exact phenotypic deficits across tissues may be related to the tissue-specific coexpression pattern and functional redundancy of different Src PTK family members. Some of the transcriptome changes may also be specific to species. However, the altered GANP expression across both avian and mammalian lymphoid tissues strengthens the physiological relevance of our findings.

Lyn PTK has a unique function in GANP-mediated proliferation

Based on our findings and previous studies, Lyn also has a unique function in B cell physiology that is conserved across different species. Changes in GANP expression in both chicken and mouse Lyn-/- models suggest that germinal center proliferation is a major and conserved function of Lyn PTK. Indeed, GANP has been previously suggested as an essential molecule for the formation of germinal centers (24). IgM and CD40 stimulation both up-regulate expression of GANP, and Lyn-/- B cells do not respond to IgM stimulation. Our data show that stimulation by CD40 also fails to induce GANP response in Lyn-/- B cells, thus confirming previous reports (47, 48). This unresponsiveness is the most likely underlying cause of the lack of proliferative response in the Lyn-deficient mice and may contribute to some of their phenotypic deficits (no Peyer’s patches, reduced number of mature B cells in spleen). Furthermore, SRBC stimulation does not lead to a proliferating population of GANP+ cells in splenic germinal centers. This absence of proliferation supports a physiological role for Lyn-dependent GANP expression as part of B cell response.

Lyn affects PU.1

We have previously shown that GANP expression is partially controlled by a critical PU.1 binding site in its promoter region (28). In this study, we show that the presence of Lyn regulates PU.1 expression and PU.1-dependent activity of the GANP promoter. Transient transfection of the GANP promoter showed it to be more active in wild-type DT40 cells than Lyn-deficient DT40 cells. Moreover, mutation of the PU.1 consensus binding site in the GANP promoter abrogated the differential response. Although no reports have yet linked PU.1 with Lyn, both share similar tissue distribution (primarily B lymphocytes and myeloid-derived granulocytes, eosinophils, macrophages, and dendritic cells) (49, 50). Further review of promoter sites of other genes identified in this Lyn microarray screen, such as CD79b and CD22 (51), revealed putative PU.1 binding sites. PU.1 expression itself is autoregulated (52), which would be consistent with our finding that PU.1 expression was decreased in Lyn-deficient DT40 cells. Furthermore, PU.1 activity is enhanced by its phosphorylation, which may be mediated by Akt or Jnk (53, 54). Both of these serine kinases are regulated by downstream events dependent on Lyn (55, 56, 57). Together, these studies demonstrate the utility of microarray analysis in identifying a small subset of physiologically relevant genes and transcription factor across species, which were not previously considered to be part of a signaling pathway. Based on these studies, we propose a Lyn->PU.1->GANP pathway in developing B cells.


   Acknowledgments
 
We thank Joshua Lovelock and Deborah Holligshead for help with cDNA array data analysis. We are grateful to Drs. Diana Linnekin and Clifford Lowell for providing Lyn-deficient mice. We are also thankful to Dr. Nina F. Schor and Dana Murphy for providing help with data verification and experiments, and for valuable comments on the manuscript.


   Footnotes
 
1 Z.K.M. was supported by the Caligiuri Fund of the Pittsburgh Foundation, the Lupus Foundation, the Bear Necessities Pediatric Cancer Foundation, and Children’s Hospital of Pittsburgh Research Advisory Committee. J.B. is supported by U.S. Department of Agriculture Grant NRICGP 98-35205-6640. K.M. is supported by National Institutes of Health Grant R21MH/NS62760-01. S.J.C. is supported by National Institutes of Health K02HL03794 and R29CA74422, and American Cancer Society Grant DHP-135. Back

2 Address correspondence and reprint requests to Dr. Seth J. Corey, Division of Pediatrics, University of Texas-M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; or Dr. Zeljka Korade Mirnics, Department of Pediatrics, University of Pittsburgh, School of Medicine, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213. Address questions regarding the microarray to Dr. Karoly Mirnics, Departments of Psychiatry and Neurobiology, PittArray Microarray Core, University of Pittsburgh, Pittsburgh, PA 15261. E-mail addresses: sjcorey@mdanderson.org, zkorade@pitt.edu, karoly{at}pitt.edu Back

3 Abbreviations used in this paper: BCR, B cell receptor; PTK, protein tyrosine kinase; GANP, germinal center-associated nuclear protein (germinal center-associated DNA primase); BDE, balanced differential expression; EF1{alpha}, elongation factor 1{alpha}. Back

Received for publication July 7, 2003. Accepted for publication February 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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